T cell selection in the thvmus Yujiro Tanaka, M.D. Labolatory of Molecular Immunology National Institute for Medical Research The Ridgeway Mill Hill London NW7 lAA UK Submitted in partial fulfilment of the requirements of the University College of London for the degree of Doctor of Philosophy April, 1995 ProQuest Number: 10044338 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10044338 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 T i t l e ...................................................... I Abbreviations .......................................... Ill A b s t r a c t .................................................. IV Acknowledgements ....................................... VI List of figures ......................................... VII List of tables ........................................... IX Index of contents ......................................... X Chapter 1 Introduction ................................ 1 Chapter 2 Materials and methods ..................... 18 Chapter 3 Transgenic oncogenesis ..................... 38 Chapter 4 TCR transgenic m i c e .......................... 68 Chapter 5 Positive selection of T cells .............. 79 Chapter 6 Discussion .................................. 96 R e f e r e n c e s ..............................................105 II Abbreviations aMEM a minimum essential medium /32m )8-2 microglobulin BM bone marrow bp base pairs ESA bovine serum albumin CDR complementality determining region CFU-S colony forming units of spleen CTL cytotoxic T lymphocyte DMEM Dulbecco's modified Eagle's essential medium DN double negative DNA deoxyribonucleic acid DP double positive Eats Ea/tsA58 EDTA ethylendiamine tetra-acetic acid FACS fluoresence activated cell sorter FCS fetal calf serum FTOC fetal thymic organ culture g gram(s) H2ts H-2K^/tsA58 HSA heat stable antigen Hyb hybridisation IFN-7 interferon-7 IMDM Iscove's modified Dulbecco's medium MHO major histocompatibility complex ml m i n i liter(s) Mis minor lymphocyte stimulating (antigen) mM mini molar NP nucleoprotein Ntg non-transgenic PBS phosphate buffered saline PGR polymerase chain reaction RAG recombination activating gene RNA ribonucleic acid RT reverse transcription SOS sodium dodecyl sulfate SP single positive SSC 3 M NaCl, 0.35 M sodium citrate ssDNA salmon sperm DNA TAE 40 mM Tris-acetate, 1 mM EDTA TBE 45 mM Tris-borate, 1 mM EDTA TCR T cell antigen receptor TE 10 mM Tris, 1 mM EDTA TES 10 mM Tris, 1 mM EDTA, 0.5 % SDS TM Thy-l/c-myc TNE 50 mM Tris, 100 mM NaCl, 100 mM EDTA III Abstract Expression of T cell antigen receptors (TCR) and their interaction with stromal ligands determine the fate of developing T cells in the thymus. To dissect molecular mechanisms involved in T cell selection, in vitro differentiation and selection system was developed using the thymocytes from a/3 TCR (F5) transgenic mice, thymic stromal cell lines derived from oncogene transgenic mice, and synthetic peptides presented by class I major histocompatibility complex (MHC). Chapter 3 describes mice carrying a temperature sensitive SV40 large T antigen under the control of class I H-2K^ promoter (H2ts mice). These mice develop normally except for the enlargement of the thymus in the adults. Conditionally immortalised thymic stromal cell lines were established from such hyperplastic thymic tissues. To compare antigen presentation capacities, thymic cortical epithelial cell lines and freshly isolated thymic dendritic cells were co-cultured with immature thymocytes or mature T cells from F5 TCR transgenic mice in the presence or absence of cognate peptide. The results show that cortical epithelial cells are as efficient as dendritic cells in negative selection of F5 thymocytes, but not in activating mature F5 T cells. In an attempt to establish cell lines which support positive selection of T cells, adherent cells in a thymic tumour of an H2ts mouse were purified using magnetic beads coated with antibodies against CD45, class II MHC, and a medullary epithelial marker. Several epithelial cell lines expressing class II H-2A*’ and a cortical marker ER-TR4 were established, and their function was assessed by reaggregate culture (chapter 5). Immature T cell lines were derived from mice which express c-myc proto-oncogene under the control of Thyl gene promoter (TM mice) . These mice develop thymic tumours consisting predominantly of CD4+CD8+ (DP) cells which are mono- or oligo-clonal. Overexpression of c-myc is associated with increased apoptosis of thymocytes in vivo, and DP cell lines derived from the tumours retained their abilities to undergo apoptosis upon TCR stimulation. However, it was not possible to induce differentiation of these DP cells to CD4 or CD8 single positive (SP) cells. Chapter 4 describes development of T cells in mice transgenic for an ajS TCR which was isolated from a cytotoxic clone F5 specific for a peptide from influenza nucleoprotein and class I H-2D^. Ontogeny of T cells in F5 mice is largely similar to that in normal mice for expression of CD4, CD8 , and TCR, except for slightly earlier appearance of immature CD8 SP cells and DP cells. The data suggest that expression of functional ajS TCR enhances transition from CD4CD8" (DN) to DP cells. Addition of cognate peptide in F5 fetal thymic organ culture causes severe block at the stage between immature CD8 SP and DP cells. A significant number of DP cells which develop in the presence of cognate peptide do not express F5 TCR and are likely to escape from cell death by expression of endogenous TCR since these do not appear in F5/RAG1^" thymic lobes. IV Chapter 5 describes effects of thymic stromal cell lines and peptide analogues on F5 T cell development in vitro. F5 TCR transgenic mice were bred to non-selecting MHC backgrounds such as H -24 or /3-2 microgrobulin (#2m)-deficient mice, in which T cell development was arrested at DP stage. Total thymocytes, including T cell progenitors and stromal cells expressing non­ selecting MHC, were then reaggregated with thymic cortical epithelial cell lines which express class I H-2^. The data show that development of F5^^ or F5/j82m'^" T cells can be restored by such epithelial cell lines. Further in an attempt to identify peptide/MHC ligands which mediate signals for positive selection of F5 T cells, peptide analogues were designed by introducing single amino acid substitutions in nonameric cognate peptide. The present study on F5 T cell development in fetal thymic organ culture confirms importance of side chains of residues at positions 4 and 7 of the peptide for interaction with TCR, as predicted from crystallographic data by others. Two of the peptides exhibit antagonistic activity and one of them appears to augment positive selection of F5 T cells if provided with suboptimal dose of cognate peptide. These data may imply significance of the presence of heterogeneous peptides in vivo for positive selection of T cells. V Acknowledgements I am deeply indebted to my supervisor, Dr. Dimitris Kioussis, for his continuous encouragement and consideration upon my work and life during my stay in London. I would also like to thank Mrs. Trisha Norton for her excellent helps in maintaining animals, Mr. Chris Atkins for assistance in sorting lymphocytes, and Drs. Clio Mamalaki, Elaine Dzierzak, and Brigitta Stockinger (NIMR, London), Drs. Farmjit Jat, Paris Ataliotis, Mark Noble (Ludwig Institute, London), Dr. Richard Flavell (Yale University, USA), Drs. Marry Ritter and Heather Ladyman (Hammersmith Hospital, London), Dr. Mark Bix (UCSF, USA), and Drs. Graham Anderson, Eric Jenkinson, and John Owen (Bermingam University, Bermingham) for their techinical assistance and helpful discussions. I would like to thank Dr. Eugenia Spanopoulou (New York University, New York) for kindly providing us with RAGl-deficient mice. Dr. Jaenish (MIT, USA) for /82m-deficient mice, Drs. Rose Zamoyska (NIMR), Anna Sponaas (NIMR), Kyuhei Tomonari (CRC, London), Brigitte Lane (Ludwig Institute, London), Marry Ritter (Hammersmith Hospital, London), Willem van Ewijk (Rotterdam University, Holland), and Erick Jenkinson (Bermingham) for antibodies. Dr. Paul Travers (ICRF, London) for making computer models of peptides bound to H-2D^ molecules. My special thanks go to Ms. Michel Burke for her secretarial service, and Mr. Mauro Tolaini, Drs. Paola Corbella and Richard Festenstein, and other members of Dr. Kioussis' and Dr. Frank Grosveld's laboratories for many exciting discussions and technical advice. This work was supported by grants from the Medical Research Council and the Leukaemia Research Fund. VI List of figures No. Contents Chapters 1 T cell development in the thymus 1 2 H-2K^/tsA58 transgenic mice 3.2.1 3 Northern blot analysis of tsA58 expression in H2ts mice 3.2.1 4 Histology of thymic tumours in H2ts6 mice 3.2.1 5 Immunohistochemistry of the thymus in CBA H2tsl mouse 3.2.1 6 Thymic and lymph node T cells in tsA58 transgenic mice 3.2.1 7 Southern blot analysis of TCR jS gene rearrangement in H2ts and TM mice 3.2.1